U.S. patent application number 13/006398 was filed with the patent office on 2011-05-05 for binding reagent system for homogeneous immunoassays for multiple allergens.
Invention is credited to CHRISTOPHER R. BROWN, James T. Murai.
Application Number | 20110104010 13/006398 |
Document ID | / |
Family ID | 21721117 |
Filed Date | 2011-05-05 |
United States Patent
Application |
20110104010 |
Kind Code |
A1 |
BROWN; CHRISTOPHER R. ; et
al. |
May 5, 2011 |
BINDING REAGENT SYSTEM FOR HOMOGENEOUS IMMUNOASSAYS FOR MULTIPLE
ALLERGENS
Abstract
A homogeneous immunoassay method and system for quantitative
determination of total immunoglobulin E and specific antibody
levels to a plurality of allergens, in which a relatively small
sampling of blood is required. The method utilizes relatively small
microparticles in aqueous suspension. The immunoassay procedure is
an immunometric sandwich procedure preferably utilizing
biotin-streptavidin signal amplification techniques and
R-phycoerytherin fluorescent labels.
Inventors: |
BROWN; CHRISTOPHER R.; (San
Mateo, CA) ; Murai; James T.; (San Bruno,
CA) |
Family ID: |
21721117 |
Appl. No.: |
13/006398 |
Filed: |
January 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12316838 |
Dec 16, 2008 |
7892853 |
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13006398 |
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11786193 |
Apr 10, 2007 |
7491553 |
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12316838 |
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10387790 |
Mar 12, 2003 |
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11786193 |
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10006483 |
Dec 6, 2001 |
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10387790 |
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Current U.S.
Class: |
422/69 ;
436/501 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 33/582 20130101; G01N 33/54313 20130101 |
Class at
Publication: |
422/69 ;
436/501 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Claims
1. A binding agent system for use in a homogeneous immunoassay
testing for an allergic response in a patient, comprising: labeled
subsets of specific allergen-coupled particles for combining with
an undiluted serum sample from the patient under conditions that
enable binding of the allergen-coupled particles to
allergen-specific IgE antibodies from the patient's serum sample,
the combining creating a response mixture; a first binding agent
for adding to the response mixture, the first binding agent
comprising an anti-human IgE antibody covalently bound to a first
member of a binding pair; a labeled second binding agent for adding
to the response mixture containing the first binding agent, the
labeled second binding agent comprising a second member of the
binding pair and a label, wherein the second member of the binding
pair has an affinity for binding to the first member of the binding
pair; and, wherein, the first binding agent and the second binding
agent are preselected to enable detection of anti-human IgE
antibodies at a detection sensitivity ranging from about 10.sup.-9
g/ml to about 10.sup.-12 g/ml, and the undiluted serum is limited
to having a volume of about 0.5 .mu.L to about 25 .mu.L; the
molecular ratio of anti-human antibody to the first member of the
binding pair in the first binding agent is from about 1:10 to about
1:30, the second binding agent has a molecular weight of between
about 400,000 and about 1,000,000 Daltons, and the selection of the
first binding agent, the second binding agent, and serum volume has
the function of avoiding quenching of a signal from the label of
the labeled second binding agent and high dose hook effects; and,
the detection sensitivity ranging from 10.sup.-9 g/ml to 10.sup.-12
g/ml is measured under homogeneous assay conditions. the amounts of
the allergen-specific antibodies from the serum sample that bound
to specific allergens are determined by (i) identifying and
distinguishing the labeled particles to identify the subsets of
specific allergens and (ii) measuring the amount of labeled second
binding agent in each of the subsets of allergens.
2. The system of claim 1, wherein the labeled particles are
fluorescently labeled microspheres, and the undiluted serum sample
ranges in volume from about 1.0 .mu.L to about 10 .mu.L.
3. The system of claim 1, wherein the first binding agent comprises
an anti-human IgE-antibody-biotin conjugate having about 15-25
biotin molecules per antibody molecule, the second binding agent
comprises a streptavidin-phycoerytherin conjugate, and the mole
ratio of the first binding agent to the second binding agent ranges
from about 1:1 to about 1:5.
4. The system of claim 1, wherein the labeled particles enable
identification of subsets of allergens, and the subsets compose a
panel of allergens having from about 2 specific allergens to about
100 specific allergens.
5. The system of claim 1, wherein the system further comprises a
kit having a device for puncturing the skin and obtaining a blood
sample, and a container for holding and transmitting the blood
sample to a laboratory for extracting the serum sample and
measuring the allergic response of the patient to an allergen or
panel of allergens.
6. The system of claim 1, wherein the system further comprises a
computer network component having testing results for the patient
on a non-transitory computer readable storage medium accessible by
the patient to obtain testing results.
7. The system of claim 1, wherein the first binding agent comprises
an anti-human antibody-digoxin conjugate.
8. A binding agent system for use in a homogeneous immunoassay
testing for an allergic response in a patient, comprising: labeled
subsets of specific allergen-coupled particles for combining with
an undiluted serum sample from the patient under conditions that
enable binding of the allergen-coupled particles to
allergen-specific antibodies from the patient's serum sample, the
combining creating a response mixture; a first binding agent for
adding to the response mixture, the first binding agent comprising
an anti-human antibody covalently bound to a first member of a
binding pair; a labeled second binding agent for adding to the
response mixture containing the first binding agent, the labeled
second binding agent comprising a second member of the binding pair
and a label, wherein the second member of the binding pair has an
affinity for binding to the first member of the binding pair; and,
wherein, the first binding agent and the second binding agent are
preselected to enable detection of anti-human antibodies at a
detection sensitivity ranging from about 10.sup.-9 g/ml to about
10.sup.-12 g/ml, and the undiluted serum is limited to having a
volume of about 0.5 .mu.L to about 25 .mu.L; the molecular ratio of
anti-human antibody to the first member of the binding pair in the
first binding agent is from about 1:10 to about 1:30, the second
binding agent has a molecular weight of between about 400,000 and
about 1,000,000 Daltons, and the selection of the first binding
agent, the second binding agent, and serum volume has the function
of avoiding quenching of a signal from the label of the labeled
second binding agent and high dose hook effects; and, the detection
sensitivity ranging from 10.sup.-9 g/ml to 10.sup.-12 g/ml is
measured under homogeneous assay conditions. the amounts of the
allergen-specific antibodies from the serum sample that bound to
specific allergens are determined by (i) identifying and
distinguishing the labeled particles to identify the subsets of
specific allergens and (ii) measuring the amount of labeled second
binding agent in each of the subsets of allergens.
9. The system of claim 8, wherein the labeled particles are
fluorescently labeled microspheres, and the undiluted serum sample
ranges in volume from about 1.0 .mu.L to about 10 .mu.L.
10. The system of claim 8, wherein the first binding agent
comprises an anti-human antibody-biotin conjugate having about
15-25 biotin molecules per antibody molecule, the second binding
agent comprises a streptavidin-phycoerytherin conjugate, and the
mole ratio of the first binding agent to the second binding agent
ranges from about 1:1 to about 1:5.
11. The system of claim 8, wherein the labeled particles enable
identification of subsets of allergens, and the subsets compose a
panel of allergens having from about 2 specific allergens to about
100 specific allergens.
12. The system of claim 8, wherein the system further comprises a
kit having a device for puncturing the skin and obtaining a blood
sample, and a container for holding and transmitting the blood
sample to a laboratory for extracting the serum sample and
measuring the allergic response of the patient to an allergen or
panel of allergens.
13. The system of claim 8, wherein the system further comprises a
computer network component having testing results for the patient
on a non-transitory computer readable storage medium accessible by
the patient to obtain testing results.
14. The system of claim 8, wherein the first binding agent
comprises an anti-human antibody-digoxin conjugate.
15. The system of claim 8, wherein the allergen-specific antibodies
from the patient's serum sample are allergen-specific IgG
antibodies, and the anti-human antibody in the first binding agent
is an anti-human IgG antibody.
16. A binding agent system for use in a homogeneous immunoassay
testing for an allergic response in a patient, comprising: labeled
subsets of specific allergen-coupled particles for combining with
an undiluted serum sample from the patient under conditions that
enable binding of the allergen-coupled particles to
allergen-specific IgG antibodies from the patient's serum sample,
the combining creating a response mixture; a first binding agent
for adding to the response mixture, the first binding agent
comprising an anti-human IgG antibody covalently bound to a first
member of a binding pair; a labeled second binding agent for adding
to the response mixture containing the first binding agent, the
labeled second binding agent comprising a second member of the
binding pair and a label, wherein the second member of the binding
pair has an affinity for binding to the first member of the binding
pair; and, wherein, the first binding agent and the second binding
agent are preselected to enable detection of anti-human IgG
antibodies at a detection sensitivity ranging from about 10.sup.-9
g/ml to about 10.sup.-12 g/ml, and the undiluted serum is limited
to having a volume of about 0.5 .mu.L to about 25 .mu.L; the
molecular ratio of anti-human antibody to the first member of the
binding pair in the first binding agent is from about 1:10 to about
1:30, the second binding agent has a molecular weight of between
about 400,000 and about 1,000,000 Daltons, and the selection of the
first binding agent, the second binding agent, and serum volume has
the function of avoiding quenching of a signal from the label of
the labeled second binding agent and high dose hook effects; and,
the detection sensitivity ranging from 10.sup.-9 g/ml to 10.sup.-12
g/ml is measured under homogeneous assay conditions. the amounts of
the allergen-specific antibodies from the serum sample that bound
to specific allergens are determined by (i) identifying and
distinguishing the labeled particles to identify the subsets of
specific allergens and (ii) measuring the amount of labeled second
binding agent in each of the subsets of allergens.
17. The system of claim 16, wherein the labeled particles are
fluorescently labeled microspheres, and the undiluted serum sample
ranges in volume from about 1.0 .mu.L to about 10 .mu.L.
18. The system of claim 16, wherein the first binding agent
comprises an anti-human IgG-antibody-biotin conjugate having about
15-25 biotin molecules per antibody molecule, the second binding
agent comprises a streptavidin-phycoerytherin conjugate, and the
mole ratio of the first binding agent to the second binding agent
ranges from about 1:1 to about 1:5.
19. The system of claim 16, wherein the labeled particles enable
identification of subsets of allergens, and the subsets compose a
panel of allergens having from about 2 specific allergens to about
100 specific allergens.
20. The system of claim 16, wherein the system further comprises a
kit having a device for puncturing the skin and obtaining a blood
sample, and a container for holding and transmitting the blood
sample to a laboratory for extracting the serum sample and
measuring the allergic response of the patient to an allergen or
panel of allergens.
21. The system of claim 16, wherein the system further comprises a
computer network component having testing results for the patient
on a non-transitory computer readable storage medium accessible by
the patient to obtain testing results.
22. The system of claim 16, wherein the first binding agent
comprises an anti-human IgG-antibody-digoxin conjugate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/316,838, filed Dec. 16, 2008, which is a continuation of
U.S. application Ser. No. 11/786,193, filed Apr. 10, 2007, which is
a continuation of U.S. application Ser. No. 10/387,790, filed Mar.
12, 2003, which is a continuation-in-part of U.S. application Ser.
No. 10/006,483, filed Dec. 6, 2001; and, each of these applications
is incorporated herein in its entirety by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a homogenous immunoassay method
and a system for determining specific antibody levels to a
multiplicity of allergens from a blood sample, or for determining
total immunoglobulins E levels in such a sample, for the purpose of
diagnosing allergy.
[0004] 2. Background and Prior Art
[0005] "Allergy" is synonymous with atopy or hypersensitivity and
is the result of an immunologically mediated reaction by
individuals to various antigenic materials, known as allergens.
People with allergies produce allergen-specific immunoglobulins,
IgE and IgG, in response to exposure to normally harmless
substances from pollens, molds, dander or foods, which are inhaled
or ingested. The generated antibodies are released to circulate in
the blood and eventually fix to specific cells in tissue. Exposure
to allergens generally results in immediate or delayed reactions,
manifested in a number of commonly identifiable symptoms, such as
sneezing, itchy eyes, runny nose and inflammation of the lungs and
nasal passages. The term "allergy" is also generally synonymous
with hay fever, rhinitis, eczema, hives, and linked to the onset of
asthma. The diagnosis of allergy involves a review of the patient
history, physical examinations and running a confirmatory
diagnostic test to identify whether the patient's symptoms are of
allergic or non-allergic origin. If allergy is responsible for the
symptoms, then the allergens responsible must be identified.
Patients with atopic or allergic diseases may be mono-sensitive to
one allergen; however, sensitization to multiple allergens is more
usual. Reactions of persons to allergens can range from the
annoying to the severe or even fatal. It therefore is desirable to
be able to determine not only whether a person has allergies, but
if so, to what allergens and to what level of severity, so that
exposure can be avoided, minimized or mitigated through
pharmacotherapeutic or immunotherapeutic methods
[0006] Confirmatory diagnostic testing may be conducted by in-vivo
skin testing, in-vivo provocation testing, or in-vitro testing for
the presence of circulating allergen-specific antibodies from blood
samples. Direct provocation, by direct inhalation or ingestion of
possible offending allergens, while relevant, is unpleasant,
possibly dangerous and cannot be performed for multiple allergens
at one sitting.
[0007] Skin testing (also referred to as skin prick testing or
scratch testing) is an in vivo procedure that involves applying an
allergen sample, or more generally a multiplicity of allergens,
directly to a patient's forearm or back via a small needle scratch
and measuring the size of the inflammatory reaction (wheal) at the
applied site on the skin. Skin prick testing is widely used, is
reliable under optimal testing conditions, can be painful, is
subject to large differences in technique and interpretations, and
cannot be used on patients taking certain drugs or patients with
skin problems. Furthermore, both provocation and skin prick in-vivo
diagnostic methods have the potential for sensitizing patients to
new allergens and, in extreme cases, eliciting a life-threatening
anaphylactic reaction upon direct exposure to the offending
allergen(s).
[0008] In vitro diagnostic testing methods directly measure
circulating levels of allergen-specific antibodies in a sample of
blood obtained from patients. These methods are generally
immunoassay procedures that are reproducible, are equivalent in
sensitivity and specificity to well conducted skin prick tests, are
unaffected by any of the factors which prevent the use of either of
the two in vivo methods, and do not cause anaphylactic events.
Immunoassay techniques capable of measuring specific antibody
levels to single allergens have been employed for many years
(Johansson, S. G. O. and Yman, L., In vitro assay for
immunoglobulin E, Coin. Rev. Allergy 6, 93-139, 1988).
Alternatively, methods that measure allergen-specific levels to a
plurality of allergens simultaneously have provided more useful
screenings of allergy as, for example, described in U.S. Pat. Nos.
4,459,360 and 5,082,768. U.S. Pat. No. 6,087,188 describes a method
of detecting an antibody in a sample using paramagnetic particles
and a chemiluminescent acridinium compound bound to avidin or
streptavidin. The method described in this patent is stated to be
useful for the detection of allergens. However, the method is
limited to detection of a single allergen in a given sample.
[0009] In the field of clinical diagnostics there is a broad
category of methods available for determining an expanding list of
clinically relevant analytes. One such category is immunoassays,
which are currently used to determine the presence or concentration
of various analytes in biological samples, both conveniently and
reliably (The Immunoassay Handbook, edited by David Wild, M
Stockton Press, 1994). Immunoassays utilize specific binding agents
to target analytes in fluids, where at least one such binding agent
is generally labeled with a variety of compounds, including
radioisotopes, enzymes and fluorescent or chemiluminescent
compounds, that can be measured by radioactive disintegrations,
enzymatic induced color-producing substrates, fluorescent output or
inhibition and chemiluminescent light output. Such specific binding
agents typically include analyte specific antibodies
(immunoglobulins) and antibody fragments, receptors, lectins, and
genetically or chemically engineered artificial antibodies. Notable
immunoassay methods include, for example, radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELIZA) (Enzyme-Immunoassay,
Edward T. Maggio, CRC Press, 1980), fluorescent immunoassay (FIA)
and chemiluminescent assays (CLA) (Luminescent Assays, Perspectives
in Endocrinology and Clinical Chemistry, Vol. 1, Mario Serio and
Mario Pazzagli, Raven Press, 1982), (Bioluminescence and
Chemiluminescense, Basic Chemistry and Analytical Applications,
Marlene, A. DeLuca and William D. McElroy, Academic Press, 1981),
(Journal of Bioluminescence, Vol. 4, M. Pazzagli, et al.,
Proceedings of the Vth International Symposium on Bioluminescence
and Chemiluminescence, Wiley, 1989), etc. Numerous method
variations and devices for performing such assays are available,
are known to those familiar with the art, and can be found in the
scientific and patent literature.
[0010] Immunoassays may be heterogeneous or homogeneous.
Heterogeneous immunoassays have been applied to both small and
large molecular weight analytes and require separation of bound
materials (to be detected or determined) from free materials (which
may interfere with that determination). Heterogeneous immunoassays
may comprise an antibody or an antigen immobilized on solid
surfaces such as plastic microtiter plates, beads, tubes, or the
like or on membrane sheets, chips and pieces of glass, nylon,
cellulose or the like (Immobilized Enzymes, Antigens, Antibodies,
and Peptides, Howard H. Weetall, Marcel Dekker, Inc., 1975). In
heterogeneous immunoassays, antigen-antibody complexes bound to the
solid phase are separated from unreacted and non-specific analyte
in solution, generally by centrifugation, filtration,
precipitation, magnetic separation or aspiration of fluids from
solid phases, followed by repeated washing of the solid phase bound
antigen-antibody complex. Of particular interest are immunometric
"sandwich" assays (Immunochemistry of Solid-Phase Immunoassay, John
E. Butler, CRC Press, 1991) which first require binding of an
immobilized antigen or antibody with the target analyte from the
biological sample. Separation of the immobilized pair and
subsequent repeated washing is followed by the introduction of a
secondary binding agent specific to the analyte, said secondary
binding agents usually being chemically conjugated with
radioisotopes, enzyme, fluorescent or chemiluminescent labels
described earlier. Secondary binding agents are typically
immunoglobulin antibodies, antibody fragments, monoclonal
antibodies or recombinant antibodies. The analyte is "sandwiched"
between the first immobilized antigen or antibody and the labeled
secondary binding agent. A subsequent separation and washing is
required to remove unbound labeled secondary binding agents. Direct
measurement of the labeled, immobilized bound complex or indirect
measurement with the use of substrates is then undertaken. It can
be appreciated by those familiar with the art of conducting solid
phase immunoassays that the procedures are laborious, time
consuming and require special equipment or devices for separating
immobilized binding agents and analytes.
[0011] Homogeneous assays are, in general, liquid phase procedures
that do not utilize antigens or antibodies that are immobilized on
solid materials. Separation and washing steps are not required. The
procedures are more commonly involved with the use of
fluorescently-labeled antigens or antibodies which upon binding
with a target analyte undergo an excitation or quenching of
fluorescence emissions, due to the close steric proximity of the
binding pair. Homogeneous methods have typically been developed for
the detection of haptens, small molecules, such as drugs, hormones
and peptides. Macromolecule analytes, such as proteins or peptides
with greater than 5000 molecular weight, usually are not determined
by homogeneous methods due to a lack of assay sensitivities. A
homogeneous method for detection of proteins was reported in U.S.
Pat. No. 5,807,675 which required chemical modifications to both
binding agents, but is limited to the detection of single
analytes.
[0012] For the diagnosis of allergy determination of total
immunoglobulin E levels is helpful, but more importantly there is a
need for a convenient and reliable immunoassay method that
simultaneously measures specific immunoglobulin antibody levels to
a panel of allergens, where one or more allergens may be
responsible for the onset of allergic symptoms. It is further
desired that the immunoassay protocol assay be easily carried out
and adaptable to automation. In vitro methods currently available
utilize heterogeneous immunoassay methods where separation and
washing is required, making them labor-intensive, time-intensive
and difficult to automate. It also would be advantageous to combine
the versatility and sensitivity of solid phase heterogeneous assay
methods with the ease of a homogeneous protocol. Furthermore,
conducting in vitro allergy tests on a panel of allergens requires
drawing a significant sample, generally 3-5 milliliters, of venous
blood from the patient. Indeed, the patient must visit a laboratory
or physician's office for the single purpose of having the blood
sample drawn.
[0013] Recent advances in immunoassay methods have introduced
microtechniques that utilize smaller solid phases and smaller
sample requirements. One example is a microimmunoassay method for
conducting analysis and detection of multiple biomolecules that is
described in U.S. Pat. No. 5,981,180. Apparatus of this general
type has been marketed by the Luminex Corporation under the
trademark FlowMetrix.RTM.. The technology incorporates a flow
cytometric procedure and the use of small, 5.6 micrometer
polystyrene bead sets, each set containing an internal fluorescent
signature, that enables detection of multiple analytes. For in
vitro allergy testing, where specific levels of antibody are in low
concentrations in circulating blood, undiluted blood or serum is
required to enable the detection of antibodies that are at
sub-nanogram- to picogram-per-milliliter levels. Up to now,
heterogeneous rather than homogeneous immunoassay methods have been
employed with existing in-vitro allergy testing methods, since
undiluted blood or serum very often contains microgram levels of
free or non-specific (total) antibody levels which can interfere
with determinations using homogeneous assays.
[0014] Homogeneous assay methods using undiluted blood or serum to
measure sub-nanogram- or picogram-per-milliliter levels of antibody
have been known to show falsely low or falsely undetectable levels
of specific antibodies in samples where the free or non-specific
antibody levels in undiluted serum are in the microgram per
milliliter range. Such false results are known as a "hook effect".
The hook effect is described by Robard, D., (Radioisotopes: 37,
(10), 1988), in the Immunoassay Handbook (Edited by David Wild, M
Stockton Press 1994) and the Manual of Clinical Laboratory
Immunology (4th ed., Rose, et al., Editors) published by the
American Society for Microbiology (Chapter 2, page 5). It involves
an unexpected fall in the amount of an analyte at the high end of a
dose-response curve, which results in a gross underestimation of
the analyte. The hook effect is caused by high concentrations of
free, i.e., unbound, analyte from neat serum or blood samples that
bind to secondary binding agents, depleting the availability of the
secondary binding agent to the solid phase bound analyte,
subsequently rendering a falsely lower signal, hence indicating a
falsely low analyte concentration. Heterogeneous assay formats,
specifically, immunometric sandwich assays, generally circumvent
the interference with the separation and washing steps inherent to
the procedures.
[0015] The above-mentioned references suggest several ways that
laboratories can deal with this hook effect. One suggested strategy
is to run all patient samples at two dilutions as a screen for this
problem. If the more dilute sample indicates a significantly higher
level of analyte, the laboratory is alerted to the possibility of a
hook effect. Various dilutions can then be carried out to provide
an accurate determination of the existing amount of analyte in the
sample. Such procedures, of course, result in duplication of work
and lengthening of the time and costs required to conduct testing
of the sample from a given patient. Alternatively, large excesses
of secondary binding agent, sufficient to bind both bound and
unbound specific and non-specific analyte antibody can be used.
However, this approach has not proved practical due to the large
concentrations of unbound specific and non-specific analyte
antibody in undiluted blood, the relatively large serum or plasma
volumes typically required in conducting in-vitro allergy tests,
and the costs for secondary binding agent that would be used.
[0016] It would be advantageous to provide a homogeneous assay
method for detecting the presence of specific antibodies to a
multiplicity of allergens, simultaneously in a single test, or
determining the total IgE antibody content in a blood sample, that
would not be prone to the occurrence of the hook effect. It also
would be advantageous to provide a method of testing blood samples
from a patient for allergies that could eliminate the requirement
that the patient visit a laboratory or physician's office, as well
as the need to have a fairly substantial amount of blood drawn for
the purpose of this test. The invention described herein provides
such advantages, as well as others that may be apparent from the
information described.
SUMMARY OF THE INVENTION
[0017] Speaking very generally, this invention provides a method
and a system for simultaneously detecting and quantifying levels of
specific immunoglobulin antibodies to a plurality of allergens,
using a very small sampling of blood, for the purpose of diagnosing
allergy. The invention also provides a method and a system for
detecting and quantifying levels of total immunoglobulin E in such
a small blood sampling.
[0018] In general, the method of this invention comprises
simultaneously measuring specific immunoglobin antibody levels to
from 2 to about 100 allergens in a blood sample, said method
comprising: [0019] (a) collecting a blood sample of from about 10
to about 100 .mu.L from an individual; [0020] (b) performing a
homogeneous immunoassay on the blood sample or a portion thereof,
wherein the immunoassay is capable of determining specific
immunoglobin antibody levels to from 2 to about 100 allergens; and
[0021] (c) determining the said specific immunoglobin antibody
levels, [0022] and the system comprises a system for simultaneously
measuring a plurality of specific immunoglobin antibody levels by
use of a homogeneous immunoassay, said system comprising: [0023]
(a) a plurality of particles coupled to from 2 to about 100
allergens; [0024] (b) a first conjugate comprising an anti-human
IgE or IgG antibody conjugated to a first member of a specific
binding pair; and [0025] (c) a second conjugate comprising a second
specific binding pair member that binds to the first specific
binding pair member; the system being capable of conducting
simultaneous quantitative determination of specific immunoglobin
antibody levels to from 2 to about 100 allergens, by a homogeneous
immunoassay.
[0026] The system and method thus enable, for the first time, the
conducting of simultaneous determinations of a plurality (from 2 to
about 100) of allergens on a relatively small sample of blood.
[0027] One preferred method comprises a homogeneous immunoassay for
simultaneously detecting and quantifying specific immunoglobulin
antibodies to a plurality of allergens in a blood sample,
comprising: [0028] (a) contacting a blood sample having a volume of
from about 1 to about 25 .mu.L, preferably 1-10 .mu.L, more
preferably 1-5 .mu.L, with a plurality of particles coupled to a
plurality of allergens, the particles being in suspension in from
about 1 to about 50 .mu.L, preferably from about 1 to about 10
.mu.L, of an aqueous medium, each combination of particles with a
specific allergen being distinguishable from combinations of the
particles with other allergens, under conditions whereby
allergen-specific immunoglobulin antibodies present in the blood
sample bind specifically to one or more of the allergens that are
coupled to the particles; [0029] (b) thereafter contacting the
materials from step (a) with a first conjugate comprising an
anti-human IgE or IgG antibody conjugated to a first member of a
specific binding pair, said specific binding pair having the
capability of amplifying fluorescent signal output, under
conditions whereby the anti-human antibody binds to the
immunoglobulin antibodies; [0030] (c) thereafter contacting the
materials from step (b) with a second conjugate containing a
fluorophore moiety coupled to a second member of the specific
binding pair under conditions whereby the second member of the
specific binding pair binds to the first member of the pair; and
[0031] (d) thereafter measuring the fluorescence emitted by the
products of step (c).
[0032] A second method comprises a homogeneous immunoassay for
detecting and quantifying total immunoglobulin E levels in a blood
sample, comprising: [0033] (a) contacting a blood sample having a
volume of from about 0.5 to about 10 .mu.L, preferably from about 1
to about 5 .mu.L, more preferably from about 1 to about 2 .mu.L,
with a plurality of particles coupled to anti-human immunoglobulin
E, the particles being in suspension in from about 1 to about 50
.mu.L of an aqueous medium; [0034] (b) thereafter contacting the
materials from step (a) with a first conjugate comprising an
anti-human IgE antibody conjugated to a first member of a specific
binding pair, said specific binding pair having the capability of
amplifying fluorescent signal output, under conditions whereby the
anti-human antibody binds to the anti-human immunoglobulin E;
[0035] (c) thereafter contacting the materials from step (b) with a
second conjugate containing a fluorophore moiety coupled to a
second member of the specific binding pair under conditions whereby
the second member of the specific binding pair binds to the first
member of the pair; and [0036] (d) thereafter measuring the
fluorescence emitted by products of step (c).
[0037] Similarly, one system of this invention comprises a system
for quantitative determination of specific immunoglobulin (IgE or
IgG) antibody levels to allergens in a blood sample comprising:
[0038] (a) a plurality of particles coupled to a plurality of
allergens, the particles being in suspension in from about 1 to
about 50 .mu.L it of an aqueous medium, each combination of
particles with a specific allergen being distinguishable from
combinations of the particles with other allergens; [0039] (b) a
first conjugate comprising an anti-human IgE or IgG antibody
conjugated to a first member of a specific binding pair, said
specific binding pair having the capability of amplifying
fluorescent signal output, the first conjugate having from 10 to 30
molecules, preferably 15 to 25 molecules, of the first binding pair
member conjugated to one molecule of the anti-human antibody;
[0040] (c) a second conjugate comprising a second member that binds
specifically to the first member of the specific binding pair, the
second member being coupled to a fluorophore moiety; [0041] (d) the
mole ratio of first conjugate to the second conjugate being from
about 1:1 to about 1:5, preferably from about 1:1 to about 1:2.
[0042] The invention further comprises a system for quantitative
determination of total immunoglobulin E levels in a blood sample
comprising: [0043] (a) a plurality of particles coupled to
anti-human IgE antibody, the particles being in suspension in from
about 1 to about 50 .mu.L of an aqueous medium; [0044] (b) a first
conjugate comprising an anti-human IgE or IgG antibody conjugated
to a first member of a specific binding pair, said specific binding
pair having the capability of amplifying fluorescent signal output,
the first conjugate having from 10 to 30 molecules, preferably 15
to 25 molecules, of the first binding pair member conjugated to one
molecule of the anti-human antibody; [0045] (c) a second conjugate
comprising a phycobiliprotein and a second specific binding pair
member that binds specifically to the first specific binding pair
member; [0046] (d) the mole ratio of the first conjugate to the
second conjugate being from about 1:1 to about 1:5, preferably from
about 1:1 to about 1:2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1 is a tabulated and graphical view of a specific IgE
assay against a panel of ten allergens according to the present
invention in accordance with Example 1
[0048] FIG. 2 is a tabulated and graphical view of a total IgE
assay in accordance with Example 2.
DETAILED DESCRIPTION OF THE INVENTION
[0049] This invention provides a method and a system for detecting
and quantifying specific antibodies to allergens in a blood sample
of an individual, for the diagnosis of allergy. Heretofore, in
vitro tests for allergy have employed immunoassay methods requiring
venous draw samplings of blood to enable testing on a panel of
allergens. Typically, sample requirements of current methods, to
determine levels of specific antibodies against a panel of five or
more allergens, are from one to three milliliters of blood. As
compared to the prior art, the invention limits the sample volume
requirements to ten microliters of sample or less, which can be
used to test against two or more allergens, preferably against ten,
twenty, forty or more allergens. The amount of sample required is
sufficiently small that individuals can remotely obtain a blood
sample using a fingerstick or similar procedure. The invention
removes the need for obtaining blood samples by venous drawing
procedures at physicians' offices, hospital laboratories or
clinical reference laboratories.
[0050] In a preferred embodiment of this invention, the individual
whose blood is to be tested is provided with a kit for collecting
and mailing, or otherwise submitting, the blood sample. The kit
typically contains one or more devices for puncturing the skin of a
finger or other portion of the body (e.g., a "fingerprick" or
"fingerstick" device) in order to draw some blood, an alcohol or
other antiseptic swab for cleaning the area to be punctured, a vial
or other small container in which the blood is to be put and
sealed, and an appropriate container for mailing the blood sample
to the laboratory for testing. The kit also may contain additional
items such as adhesives to protect the wound, instructional inserts
and labels with individual identification numbers (e.g., PIN) for
samples. In any case, the exact form and contents of such a kit can
vary according to the desires of the laboratory and do not form a
part of this invention. Numerous components for such kits are
readily available for collection and/or testing of blood (for
instance, at home) for various purposes.
[0051] In addition, it should be noted that the providing of a
blood sample by the individual without visiting a physician's
office or laboratory is not a limiting feature of this invention,
but only an advantage of it. The blood sample can well be taken
during a visit to a physician's office, and submitted to a
laboratory for testing. In such a case, other advantages of the
invention, such as the ability to test a small sample without
requiring dilution and/or duplication of tests, are achieved.
[0052] The use of such a limited sample volume essentially avoids
the presence of large amounts of free IgE antibody in the assay and
the resulting hook effects that have heretofore been a barrier to
homogeneous formats with in vitro tests for allergy. In vitro
testing methods for diagnosing allergy have heretofore employed
heterogenous immunoassay methods, more generally, two-site
immunometric sandwich assay procedures that require separation and
washing steps to be performed. Neat serum, plasma or blood samples
are also necessary in these procedures to attain the levels of
sensitivity required to determine blood circulating levels of
specific antibody that are in the 10.sup.-9 to 10.sup.-12 molar
levels or in low nanogram/mL to picogram/mL range. Up till now
homogeneous assays have not been employed, due to high levels of
specific and nonspecific antibody that are generally present in
neat samples of blood from allergic individuals, and the occurrence
of the high-dose hook effect in these assays. The invention
provides an assay method that combines a solid phase sandwich
immunoassay procedure with a homogeneous assay format, where there
is no requirement for separation of materials, washing, etc., and
no requirement for sample dilution.
[0053] The assay method quantitates levels of total IgE and
specific antibody levels that are essential to allergy diagnosis
and to the degree or severity of allergic response to known
allergens.
[0054] A preliminary step in carrying out the method and assembling
the system for use in this invention is the preparation and
collection of a plurality of solid particles coupled to a plurality
of allergens so as to provide a panel of allergens to be tested,
for instance, to determine whether an individual's allergic
symptoms are mediated by one or more of the allergens under
investigation.
[0055] Each combination of particles with a specific allergen in
the panel is distinguishable from combinations of particles with
other allergens. A specific allergen can be comprised of extracted
proteins, protein fractions purified by chromatographic or affinity
chromatographic methods, recombinant proteins or combinations of
the above. Allergen mixes, such as two or more grasses, trees or
foods, for example, can also be utilized. The capability to
distinguish between combinations of particles with different
allergens is accomplished by providing a plurality of particles of
different types. That is, the particles may be divided into
subsets, with each subset being distinguishable from other subsets
according to a particular property, characteristic or
characteristics. For example, the particles may be divided into
subsets where each subset is capable of being distinguished by a
specific color or emission spectra, which may be provided by the
presence of a fluorochrome or combinations of fluorochromes
incorporated within or on it, for example, as described in U.S.
Pat. No. 5,981,180. Each subset of the particles is coupled to a
specific allergen so that, again, the combination of particles with
specific allergen is distinguishable from combinations of particles
with other allergens, in accordance with the particular
characteristic or characteristics that distinguishes the particle
or bead in question from others that are used. The coupling of the
allergens to the beads or particles is accomplished by covalent
coupling or adsorption methods well known to those familiar with
the art and described in the patent and scientific literature (see,
for instance, Immunochemistry of Solid--Phase Immunoassay, John E.
Butler, CRC Press, 1991 and Immobilized Enzymes, Antigens,
Antibodies, and Peptides, edited by Howard H. Weetall, Marcel
Dekker, Inc. New York, 1975).
[0056] The particles themselves are typically spherical (i.e.,
"beads or microspheres"), with either a rough or a smooth surface,
and are prepared as known in the art. They are made various
materials, usually non-porous glass, polystyrene, latex or other
polymeric materials, and are generally 0.05 micron to 90 micron
diameter, preferably 0.5 to 10 micron in diameter, with densities
ranging from about 1 to 2 g/mL, preferably close to the density of
water.
[0057] The particle/allergen combinations are preferably stored in
a buffered solution containing a protein stabilizer and a
bacteriostatic agent, for use as desired.
[0058] Once the subsets of individual allergen/particles
combinations are prepared, the subsets can be used individually or
can be mixed together to form a single assemblage of allergens
coupled to particles. This may be done either in advance of
conducting assays, so as to provide a preassembled panel of
allergens for use in general, or may be done for each individual
assay, for instance, in case it is desired to tailor one or more
assays to the geographical location or environment of certain
patients. Particle concentrations can be determined and adjusted
using a conventional counter and the desired number of particles
for the assay can be aliquoted. It is preferred that the number of
particles in suspension be of limited quantity and in a limited
volume to insure assay sensitivity, speed and adaptability to
automated microassay formats (if an automated assay is to be used).
The particles in suspension should be at a density close to that of
water and suspended in a buffered or aqueous medium sufficient in
volume to allow molecular motion and molecular contact and to be
adaptable to microassay formats.
[0059] The panel for testing can contain, overall, as few as 2,
preferably as few as about 5, and up to about 100, specific
allergens or mixtures of allergens; however, panels of 10 to 40
allergens are more common. It is preferred that for each test, the
sample is added to a suspension containing from about 1000
particles to about 4000 particles of each set of particle-coupled
allergen or mixture of allergens, preferably in a small volume, for
example, a volume of from about 1 to about 50 .mu.L, preferably
from about 5-25 .mu.L. Thus, for example, a test with a panel of 10
allergens would contain about 10,000 particles in 5-20 microliters
of a buffered solution. Likewise a test with a panel of 20
allergens would incorporate about 20,000 particles in a 5-20
microliter volume. Increasing the number of particles in an assay
decreases the amount of time required to identify and read the
particles.
[0060] The particles thus prepared are contacted with an
individual's blood sample. The blood sample will have a volume of
from about 1 to about 25 .mu.L, preferably from about 1 to about 10
.mu.L, and most preferably from about 1 to about 5 .mu.L. Those
familiar with the art will recognize that binding of analytes to
antigens or antibodies is influenced by incubation conditions such
as time, temperature, pH, ionic strength of reagents, and the like,
and the conditions of a given assay will be chosen as known in the
art to optimize the sensitivity and specificity of the test and
generally suit the ease of use of the protocol and its adaptability
to automation. In general, such conditions include a temperature of
from about 18 to about 37.degree. C., preferably ambient
temperatures of from about 18 to about 25.degree. C., and a time of
from about 15 minutes to about 24 hours, preferably 1 hour or less.
During the first incubation phase, specific antibodies from an
individual's blood sample will bind to specific allergens on
particles through normal antigen-antibody binding forces. The
suspension will contain both free specific and free non-specific
antibodies as well as particle-bound specific antibodies.
[0061] For the determination of total immunoglobulin antibodies in
a sample of blood, anti-human antibody coupled to particles are
contacted with an individual's sample. The sample will have a
volume of from about 0.5 to about 10 .mu.L, preferably from about 1
to about 5 .mu.L, most preferably from about 1 to about 2 .mu.L.
Incubation times and conditions are determined as described herein.
In one embodiment of this invention, the test for total
immunoglobins is capable of determining levels up to 4800 ng/mL
total immunoglobins.
[0062] Then, a first conjugate comprising an antibody to the
specific analyte, such as anti-human IgE, that is coupled to the
first member of a specific binding pair is sequentially added to
the mixture. Separation of the solid phase particles from the
reaction medium and sample is not required. The specific binding
pair is a pair of molecules that have binding specificity for one
another. Examples of types of specific binding pairs are
biotin-avidin, biotin-streptavidin, digoxin-antidigoxin, and
complementary homopolynucleotides poly (dA)-poly (dT) (described in
U.S. Pat. No. 6,245,513). The biotin-streptavidin specific binding
pair is a preferred embodiment of this invention. In a preferred
embodiment a plurality of one member of the specific binding pair
(e.g., biotin) is coupled (conjugated) to the antibody (e.g.,
anti-human IgE). The second member of the specific binding pair,
e.g., streptavidin, is coupled to a fluorescent label or
fluorophore moiety, as described below. The binding of the second
specific binding pair member, streptavidin, to the plurality of
biotins conjugated to antibody, amplifies the fluorescent signal,
providing greater assay sensitivity. Biotinylation of antibody can
be carried out by techniques well known in the art. Typically,
antibody-biotin conjugates either obtained commercially or prepared
contain 5 to 10 biotin molecules per antibody molecule. In this
preferred application, optimum assay sensitivity is obtained when
from about 10 to about 30 biotin molecules are coupled per antibody
molecule, preferably, about 15-25 biotin molecules per antibody
molecule.
[0063] Here again, those familiar with the art will recognize that
binding of analytes to antigens or antibodies is influenced by
incubation conditions such as time, temperature, pH, ionic strength
of reagents, and the like, and the conditions of a given assay will
be chosen as known in the art to optimize the sensitivity and
specificity of the test and generally suit the ease of use of the
protocol and its adaptability to automation. In general, such
conditions include a temperature of from about 18 to about
37.degree. C., preferably ambient temperatures of from about 18 to
about 25.degree. C., and a time of from about 15 minutes to about
24 hours, preferably 1 hour or less. During the second incubation
phase, secondary binding agents will bind to the analyte-particle
complex through normal antigen-antibody binding forces.
[0064] Selection of the conjugates is done with an objective of
enhancing assay performance and sensitivity through a 10.sup.-9 to
10.sup.-12 molar range without encountering fluorescent quenching
and hook effects.
[0065] After a suitable incubation period of the first conjugate
with the particle and sample mixture, a second conjugate,
containing the second member of the specific binding pair
conjugated to a fluorophore, is added to the mixture. The second
binding pair member is preferably avidin or streptavidin, most
preferably streptavidin, though, as mentioned above, it may be
anti-digoxin or materials described in U.S. Pat. No. 6,245,513.
Covalent attachment of fluorescent labels to avidin or streptavidin
may be effected by a variety of techniques previously described in
patent and scientific literature (Haugland, R. P., Bhalagat, M. K.,
Preparation of avidin conjugates, Methods Mol. Biol. 1998;
80:185-96). Typical fluorescent moieties are described in Chapter 3
of the Manual of Clinical Laboratory Immunology, supra.
Alternatively the conjugates may be obtained from a commercial
sources. Fluorescent dyes such as fluorescein, the arylsulfonate
cyanine dyes, phycobiliprotein dyes, bodipy dyes and the like, may
be used. If the particle subsets are distinguished from one another
on the basis of incorporation of fluorochromes, the dyes used in
the conjugates are selected so as to have fluorescent emissions
that are distinct from, and do not interfere with, the emission
spectra of the particle subsets. A preferred type of fluorescent
material is a class of compounds known as phycobiliproteins, more
particularly the phycoerytherins, the phycocyanins, and the
allophycocyanins, most preferably the phycoerytherins.
Phycoerytherin conjugates have sensitivities ranging from five to
ten times greater than that of corresponding fluorescein
conjugates, with quantum yields of up to 0.98 and extinction
coefficients of up to 2.4 million (cm.sup.-1 M.sup.-1). Most
preferred of these is R-phycoerytherin. In the second conjugate,
the molecular ratio of fluorescent dye to second binding pair
member generally can range from about 1:1 to up to about greater
than 5:10 with molecular weights ranging from about 300,000 to
3,000,000 or more daltons. The preferred molecular weight of the
second conjugate generally is about 400,000 to about 1,000,000
daltons.
[0066] The materials are incubated under appropriate conditions for
binding of the binding pair members. These, as are known in the
art, typically include a temperature of from about 18.degree. C. to
about 45.degree. C., preferably from about 18.degree. C. to about
25.degree. C. and a time of from about 15 minutes to about 24
hours, preferably from about 15 minutes to about 3 hours.
[0067] In this overall process, the fluorescently labeled
antibodies bind to the particles through the first and second
binding partners, and through the binding of antibody to analyte
antigen. They thus can be detected and measured by application of
excitation energy having a wavelength selected to excite the chosen
fluorescent label, where the emission spectra that is generated is
distinct from the emission spectra incorporated in the
particles.
[0068] An important feature of the overall method of this invention
is that the immunoassay can be conducted without sample dilution.
Furthermore, by enabling the use of small volume samples, a
homogeneous assay format with solid phase particles, with
appropriate assay range and sensitivity, is now possible.
[0069] In one embodiment of this invention, applicable to both
specific and total immunoglobin assays, the individual who
submitted the sample, or another individual acting with his or her
permission, can obtain access to the test results over the
Internet, or a similar global computer system, for instance, from a
Web site via a Web server.
[0070] As used herein, the term "Web server" may also refer to a
plurality of servers organized to handle a large number of requests
for a Web server, i.e., a distributed Web server system. The term
"Web site" is often used to refer to a collection of Web servers
organized by a business entity or other entity for their purposes.
A user is said to "go to" or "access" a Web site when the user
directs his or her Web client to make a request of one or the
site's Web servers and display the response to the user (even
though the user and the Web client do not actually move
physically). The user perception is that there is a location on the
Web where this Web site exists, but it should be understood that
the term "Web site" often refers to the Web server or servers that
respond to requests from Web clients, even though "site" does not
necessarily refer to the physical location of the Web servers. In
fact, in many cases, the servers that serve up a Web site might be
distributed physically to avoid downtime when local outages of
power or network service occur.
[0071] The term "Web site" typically refers to a collection of
pages maintained by a common maintainer for presentation to
visitors, whether the collection is maintained on one physical
server at one physical location or is distributed over many
locations and/or servers. The pages (or the data/program code
needed to generate the pages dynamically) need not be created by
the common maintainer of the collection of pages. Such a maintainer
of the collection of pages is typically referred to as the Web site
operator.
[0072] The term "Web site" also includes Web sites connected to the
Web clients via an intranet, wireless access protocol (WAP)
network, a local area network (LAN), a wide area network (WAN), a
virtual private network (VPN) or another network arrangement. "Web"
typically refers to "World Wide Web" (or just "the WWW"), a name
given to the collection of hyperlinked documents accessible over
the Internet using HTTP. As used herein, "Web" might refer to the
World Wide Web, a subset of the World Wide Web, a local collection
of hyperlinked pages, or the like.
[0073] In this embodiment of the invention, the submitter of the
sample (or someone else having authorization from the submitter,
such as a relative, physician or other care provider) can obtain
access to information respecting the test results, on a Web site,
Web "page" or the like. A Web page typically consists of certain
information. Web pages include both static pages and dynamic pages.
Static pages are pages that are stored on the server, or in storage
accessible by the server, prior to the request and are sent from
storage to the client in response to a request for that page.
Dynamic pages are pages that are generated, in whole or in part,
upon receipt of a request. For example, where the page is a view of
data from a database, a server might generate the page dynamically
using rules or templates and data from the database where the
particular data used depends on the particular request made.
[0074] The submitter can be assigned a password or code (PIN) that
permits access to his or her test results on a web page or within a
database that is maintained by, or for, the testing organization
(e.g. testing laboratory). Thus, in this embodiment a submitter may
also receive the test results without having to visit a physician's
office, and may then provide such information to a physician of his
or her choice, at his or her convenience.
[0075] Computer software that may be used to maintain, and provide
access to, such test results, is available from vendors, and does
not form a part of this invention.
[0076] The following assay procedure was used for all the examples
described herein.
[0077] a. Extracted allergens, typically from trees, grasses,
molds, foods, danders and the like, are coupled to microparticles
by covalent or adsorbed procedures. Each allergens extract is
coupled to a unique subset of particles, with each subset
distinguishable by an incorporated fluorescent emissions or
particle size where such differing characteristics can be
recognized by appropriate instrumentation. The allergen-coupled
particle subsets are stored individually or the subsets are
combined as a panel of allergens in a buffered medium with protein
stabilizers and bacteriostatic agents. To determine specific IgE
levels to a panel of 10 allergens, for example, 10 .mu.L of the
combined particle subsets, totaling 10,000 particles, each allergen
particle subset represented by 1,000 particles, are placed in a
microfuge tube or microtiter well. For total immunoglobulin
determinations, anti-human IgE is coupled to microparticles by
covalent or adsorbed procedures, and 1,000 particles are placed in
a microfuge tube or microtiter well for each test.
[0078] b. Blood, plasma or serum collected from an individual by
means of the fingerstick collection kit as described herein, or
other collection methods, is appropriately labeled or bar coded for
identification. A few drops of collected blood, plasma or serum is
adequate for determining specific antibody levels to a panel of
allergens. A 5 .mu.L sample is added to the allergen-coupled
particles residing in the microfuge tubes or microtiter wells. The
combined mixture can be briefly vortexed (with microfuge tubes) or
shaken (microtiter plate shaker) and incubated for 1 hour at room
temperature. For total immunoglobulin determinations, a 1 .mu.L
sample is added to the anti-human IgE coupled particles residing in
the microfuge tube or microtiter well for each test.
[0079] c. Then, a volume of an antibody-biotin conjugate, as
described, in a buffered solution, is added to the sample and the
allergen-coupled particle mixture described above. The total
material is then briefly vortexed or shaken again and allowed to
incubate for 1 hour at ambient temperature.
[0080] d. A volume of a streptavidin-R-phycoerytherin conjugate, as
described, in a buffered solution, is then added to the mixture
(c), and the resulting mixture is again briefly vortexed or shaken
and allowed to incubate for 1 hour at ambient temperature.
[0081] Following this incubation, the resulting mixture of
materials is subjected to a reading via appropriate flow cytometric
instrumentation, such as the Luminex Corporation Luminex 100
System.RTM. or the Becton-Dickinson Immunosytometry FACSCAN.RTM.
instruments, which simultaneously determines the particle subset by
the particle incorporated fluorescent emissions or particle size
and measures the emission spectra of the R-phycoerytherin attached
to the particle via antigen-antibody and biotin-streptavidin
binding as previously described. To determine quantitative
concentration levels of specific antibody to allergens, known
standards, traceable to the World Health Organization standard
(e.g., WHO IgE Standard 75/502 IU/mL), are run to provide a
standard curve, from which the concentration of specific IgE in a
patient's sample for each allergen is extrapolated, recorded and
reported. Specific levels of antibody to allergens can be reported
in concentration units, as for example, Units per milliliter and/or
class levels, as those acquainted with in vitro allergy diagnostics
will recognize.
[0082] The following are illustrative examples of the
invention.
Preparation of Biotinylated Antibodies:
Biotinylated Anti-Human IgE:
[0083] To 15 .mu.L of biotin (biotin amidocaproate
N-hydroxysuccinimide ester) (Sigma, Mo. USA) 25 mg/mL in
Dimethylformamide (Sigma) is added 0.4 mL of affinity purified
anti-human IgE (Bethy Labs, TX, USA) 3.0 mg/mL in 0.1M NaHCO.sub.3.
The reagent solution is stirred for 1 hour at 25.degree. C.
Hyroxylamine (Spectrum Quality Products, CA, USA), 20 .mu.L, 20
mg/mL in 0.1M NaHCO.sub.3 is added and the solution briefly mixed.
The biotinylated antibody solution is purified on a Sephadex G-25
(Pharmacia, Sweden) desalting column equilibrated with Phosphate
Buffered Saline (PBS), pH 7.2, containing 0.1% Sodium Azide
(Sigma)
Covalent Coupling of Allergens to Particles--General Procedure
[0084] Carboxylated polystyrene microspheres (Luminex Corporation,
TX, USA) are suspended in 80 .mu.L of 0.1M MES Buffer (Fisher
Scientific, PA, USA) pH 6.1. To the suspension is added 10 .mu.L of
N-Hydroxysulfosuccinimide sodium salt (Pierce Chemicals, IL, USA)
50 mg/mL and 10 .mu.L of
1-(3-dimethylaminopropyl)-3-Ethyl-carbodiimide hydrochloride
(Pierce Chemicals, IL, USA) 5 mg/mL. The solution is vortexed and
let stand in the dark for 15-20 minutes at ambient temperature. The
activated microspheres are centrifuged for 5 minutes at 5000 g and
the supernatant is aspirated. The microspheres are then resuspended
in 250 .mu.L of 0.1M PBS solution (0.1 M Sodium Phosphate, 0.14 M
NaCl) pH 7.3. The mixture is vortexed, the activated microspheres
centrifuged and the supernatant aspirated as before.
[0085] The activated microspheres are then resuspended in 250 .mu.L
of allergen extract, purified proteins or recombinant proteins.
Then the mixture is rotated (protected from light) for at least 1
hour at ambient temperature and centrifuged for 5 minutes at 5000
g. The supernatant is aspirated and the microspheres resuspended in
250 .mu.L of PBS/HSA Buffer pH 7.3, containing 0.01 M PBS and 0.02
mg/mL Human Serum Albumin (Sigma, Mo., USA), and vortexed to wash
the microspheres of residual allergen extract. The supernatant is
again centrifuged and aspirated as previously described. The wash
procedure is repeated two more times and the microspheres
resuspended in 250 .mu.L PBS/HSA. Microsphere concentrations in
solution is determined by counting using a hemocytometer (Fisher
Scientific, PA, USA) and microscope (Nippon Kogaku, Japan).
Example 1
Assay for Specific IgE Levels to Allergens
[0086] Subsets of fluorescently labeled microspheres obtained from
Luminex Corporation were covalently coupled with Alternaria mold,
Bermuda grass, Timothy grass, Cat dander, Mountain Cedar tree, Egg
white, Milk, Wheat, Ragweed and Mite extracted proteins. Serum
samples obtained from atopic and non-atopic individuals were
assayed against this panel of ten allergens. A 5 .mu.L sample was
added to 10 .mu.L of suspended allergen-coupled microspheres placed
in V-bottom polypropylene microtiter wells (Evergreen Scientific,
CA, USA) or 0.5 mL polypropylene microfuge tubes (Evergreen
Scientific, CA, USA). For the six point standard curve, 5 .mu.L of
serum standards (secondary standards calibrated against the WHO IgE
Standard 75/502) were each added to 10 .mu.L of antigen-coated
microspheres in microtiter wells. The microtiter plate was placed
on a microtiter plate shaker (Fisher Scientific, PA, USA) and
shaken for 10-20 seconds. Microspheres with standards or sample
were incubated for 1 hour at room temperature as described above. A
40 .mu.L volume of anti-human IgE-biotin conjugate was added
directly to the contents in each well; the plate was briefly shaken
as before and the contents incubated for 1 hour at ambient
temperature. Subsequently, a 40 .mu.l volume of
streptavidin-R-phycoerytherin conjugate (Advanced Biosystems, CA,
USA) was added to the contents of each well and incubated for 1
hour at ambient temperature. The microspheres were then directly
introduced to the Luminex 100 Flow cytometer for aspiration into
the instrument which simultaneously identifies the emission signals
of the subset particles and the presence of the R-phycoerytherin
fluorescent label residing on the particles. The standard curve was
determined using a four parameter Lorentizian cumulative non-linear
transition function equation and levels of specific IgE to
allergens interpolated from the standard curve as shown in FIG.
1.
[0087] FIG. 1 shows the results from two non-allergic and three
allergic individuals with positive specific IgE responses to a
panel of ten allergens. A 5 .mu.L volume of sample from each
individual was used in a three-hour homogeneous assay procedure to
simultaneously measure specific IgE levels to the ten allergens
coupled to microspheres. Measurement of fluorescent intensity
signals using the Luminex 100 System was used to report the results
in either IU/mL or class units as interpolated from a six-point
standard curve from 0.35 IU/mL. Response interpretations range from
Negative to Extremely High based on class levels obtained.
Example 2
Total IgE Assay
[0088] Goat anti-human IgE (B ethyl Laboratories, TX, USA) was
coupled to microsphere particles (Luminex Corporation, TX, USA)
following the same procedure described above for coupling
allergens. Serum samples were obtained from individuals obtained by
the methods described above. A 1.0 .mu.L volume of standard
(secondary standards calibrated against the WHO IgE Standard
75/502) or individual sample was added to 25 .mu.L of suspended
microspheres coupled with antibody in microtiter wells, briefly
shaken as described above and incubated for 1 hour. A 40 .mu.L
volume of anti-human IgE-biotin conjugate was subsequently added to
the contents of each well and incubated for 1 hour. Subsequently, a
40 .mu.L volume of streptavidin-Riphycoerytherin conjugate was
added to the contents of each well, the mictrotiter plate briefly
shaken and the mixture incubated for 1 hour. The microspheres were
then directly introduced to the Luminex 100 Flow cytometer for
aspiration into the instrument which simultaneously identifies the
emission signals of the subset particles and the presence of the
R-phycoerytherin fluorescent label residing on the particles. The
standard curve was determined using a four parameter Lorentizian
cumulative non-linear transition function equation and levels of
total IgE in the samples interpolated from the standard curve as
shown in FIG. 2.
[0089] FIG. 2 shows the results of the Total IgE Standard Curve and
Total IgE results of ten individuals. A 1 .mu.L volume of sample
from each individual was used in a three-hour homogeneous assay
procedure. The amount of total IgE bound to anti-human IgE-coupled
microspheres was determined by measuring the signal intensity from
the fluorescent reporter signals using the Luminex 100 System and
interpolated from a six-point standard curve from 2-2000 IU/mL.
Expected results on samples were provided by a reference laboratory
using an alternative essay procedure.
* * * * *